PROJECT SUMMARY Replication of the genome is a fundamental process necessary for life. During the replication of the millions of DNA base-pairs that make up a genome, the replication fork encounters barriers that must be overcome, including DNA structures, strongly bound proteins, or damaged or cross-linked bases. After the barrier is resolved or bypassed, the replication fork must restart as accurately as possible to avoid chromosome breakage; one mechanism by which this happens involves proteins that are normally used for a process called homologous recombination, which occurs by DNA strand invasion. Recently, it has been recognized that this process may be regulated by nuclear location, with the stalled replication fork relocating to the nuclear periphery to undergo restart. The long-term objective of this application is to understand the process of fork restart, its regulation by nuclear location, and its role in preventing genome instability and deleterious chromosome breaks. The specific aims are (1) to elucidate the role of nuclear pore localization in fork restart and regulation of recombination, and (2) to investigate the effect of fork restart on repeat instability. We have shown that expanded CAG repeat sequences, which form DNA structures, are particularly reliant on fork restart to prevent copy number changes and chromosome fragility. Thus it is of critical importance to study restart at structure-forming repeats as they are hotspots for mutation in the genome, and their expansion can cause degenerative diseases such as Huntington's disease, myotonic dystrophy, and ALS. In addition, structure-forming repeats are fragile sites that can break and provoke deletion of vital DNA sequences during early steps of cancer initiation, which is exacerbated by replication stress occurring in cancer cells. The outcome of this research will be a better understanding of cellular mechanisms used to cope with replication stress, and how these mechanisms can go wrong to cause genetic mutation. This information should provide insights into how to bolster or interfere with these processes, to either prevent mutation or target mutated cells for elimination.